RESUMO
Vascular endothelial growth factor receptor-3 (VEGFR-3) and its ligands, vascular endothelial growth factor-C (VEGF-C) and -D (VEGF-D), are the major molecules involved in developmental and pathological lymphangiogenesis. Here we describe for the first time the development of a specific indirect enzyme-linked immunosorbent assay (ELISA) for the quantification of VEGFR-3 in different human cell and tissue lysates. A combination of the goat polyclonal anti-VEGFR-3 antibody and the mouse monoclonal anti-human VEGFR-3 antibody was used. The assay was highly sensitive and reproducible with a detection range of 0.2-25 ng/ml. The assay was specific for VEGFR-3, with no cross-reactivity to VEGFR-1 or VEGFR-2. Complex formation with VEGF-C and VEGF-D had no effect on the sensitivity of the assay. The VEGFR-3 concentration in the lysates of cultured human dermal microvascular endothelial cells was 14-fold higher than in the lysates from human umbilical vein endothelial cells. In human kidney, breast, colon, gastric and lung cancer tissues the protein levels of VEGFR-3 were in the range of 0.6-16.7 ng/mg protein. Importantly, the level of VEGFR-3 protein detected in the ELISA correlated significantly with the number of VEGFR-3 positive vessels observed in histochemical sections, suggesting that the ELISA assay may be a reliable surrogate of measuring VEGFR-3-positive vessel density. The protein levels of VEGFR-3 in 27 renal cell carcinoma samples had a significant correlation with the levels of VEGF-C (p<0.001), or biological active, free VEGF-A (p<0.0001), but not with VEGFR-1 or total VEGF-A. This assay provides a useful tool for the investigations of the expression levels of VEGFR-3 in physiological and pathological processes, particular in cancer and in lymphangiogenesis-related disease.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Neoplasias/química , Neoplasias/patologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/análise , Animais , Anticorpos Monoclonais , Células Endoteliais , Cabras , Humanos , Camundongos , Neovascularização Patológica , Sensibilidade e Especificidade , Distribuição Tecidual , Células Tumorais Cultivadas , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/imunologiaRESUMO
Lymphangiogenesis plays an important role in several normal and pathological conditions such as wound healing, inflammation or metastasis formation in several malignancies. VEGF-C and VEGF-D are important and specific regulatory factors for lymphatic endothelial proliferation and lymphangiogenesis. In order to develop a highly sensitive and specific detection system for VEGF-C, we produced soluble binding proteins and antibodies for a microtiterplate-based assay. Here we describe a specific enzyme-linked immunosorbent assay (ELISA) for the measurement of human, rat and murine VEGF-C. The different antibodies developed against human and rat VEGF-C could be combined to detect processed and partially processed VEGF-C in a specific way. The ELISA was able to detect human and rat VEGF-C with a minimum detection limit of 100 pg/ml. The assay did not show any cross-reactivity with the related protein VEGF-D. Furthermore, complex formation with its soluble receptors VEGFR-2 and VEGFR-3 did not restricted the sensitivity of the assay. Using this assay, VEGF-C was measured in supernatants and lysates of different cell types and in tumour tissue samples of murine, rat and human origin. Cell lines secrete VEGF-C in very low amounts (<1 ng/ml) whereas VEGF-C transfected cells can secrete up to 50 ng/ml VEGF-C into the supernatant. In human tumour tissue samples VEGF-C was detected in some carcinomas in the low protein range. This ELISA will be a useful tool for investigations concerning the physiological function of VEGF-C in lymphangiogenesis under normal and pathophysiological conditions.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Fator C de Crescimento do Endotélio Vascular/análise , Animais , Anticorpos/análise , Northern Blotting , Reações Cruzadas , Humanos , Neoplasias/química , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
Eschericha coli was genetically engineered to produce recombinant human bone morphogenetic protein-2 (rhBMP-2) in a non-active aggregated form using a temperature-inducible expression system. High concentrations of both biomass (75 g cell dry weight per liter of culture broth) and inactive rhBMP-2 (8.6 gl(-1)) were obtained by applying a high-cell-density cultivation procedure. After washing and solubilizing the inclusion bodies, rhBMP-2 was refolded and dimerized at concentrations up to 100 mgl(-1) by means of a simple dilution method with yields exceeding 50%. Finally, a one-step purification procedure based on affinity chromatography was implemented to isolate the rhBMP-2 dimer. With the established renaturation and purification protocols, yields of more than 10 mg rhBMP-2 dimer per gram cell dry weight were obtained corresponding to 750 mg rhBMP-2 dimer per liter of culture broth. The purified rhBMP-2 dimer showed biological activity equivalent to CHO produced rhBMP-2 as tested by the induction of alkaline phosphatase activity in C2C12 cells.